Growth factors adsorbed on polyglycolic acid mesh augment growth of bioengineered intestinal neomucosa.

Production of tissue engineered small intestine (TESI) has been limited by the relatively large amount of native tissue required to generate neomucosa. The influence of growth factors and three-dimensional (3D) extracellular matrices on TESI has been studied both in vitro and in vivo, and positive growth effects on tissue mass and differentiation were noted. The present study investigates the impact of single doses of glucagon-like peptide-2 (GLP-2), hepatocyte growth factor (HGF), or holo-transferrin adsorbed onto a polyglycolic (PGA) mesh scaffold using a rat small-intestinal organoid transplant model. In Experiment I, intestinal organoids were seeded onto PGA mesh discs, suspended in either Matrigel (n=8) or a vehicle control (n=8), and implanted into syngenic recipients. In Experiment II, GLP-2 (n=8), HGF (n=8), or transferrin (n=8) were adsorbed onto PGA mesh discs. Intestinal organoids were then suspended in Matrigel and seeded onto each growth factor-loaded PGA disc or onto control discs without growth factors (n=12). In addition, organoids were suspended in vehicle and seeded onto control discs (n=12). All discs were implanted into syngenic recipients. After 4 wk, histologic analysis of the samples revealed significantly greater neomucosal surface area (3.62±0.33 mm(2)versus 0.92±0.11 mm(2), P<0.0001) and cyst diameter (2.83±0.14 mm versus 2.06±0.07 mm, P<0.0001) in groups treated with Matrigel compared with vehicle controls. The addition of holo-transferrin to the scaffolds further augmented neomucosal surface area (9.11±0.66 mm(2)versus 3.01±0.22 mm(2), P<0.01), whereas that of GLP-2 stimulated the formation of increased numbers of cysts (8.88±0.46 versus 4.18±0.25, P<0.01). These data suggest that Matrigel and growth factors adsorbed to polymer scaffolds can be used to manipulate the morphology of TESI.

Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo.

Oxysterols, naturally occurring cholesterol oxidation products, can induce osteoblast differentiation. Here, we investigated short-term 22(S)-hydroxycholesterol + 20(S)-hydroxycholesterol (SS) exposure on osteoblastic differentiation of marrow stromal cells. We further explored oxysterol ability to promote bone healing in vivo. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, mineralization, and Runx2 DNA binding activity. To explore the effects of osteogenic oxysterols in vivo, we utilized the critical-sized rat calvarial defect model. Poly(lactic-co-glycolic acid) (PLGA) scaffolds alone or coated with 140 ng (low dose) or 1400 ng (high dose) oxysterol cocktail were implanted into the defects. Rats were sacrificed at 6 weeks and examined by three-dimensional (3D) microcomputed tomography (microCT). Bone volume (BV), total volume (TV), and BV/TV ratio were measured. Culture exposure to SS for 10 min significantly increased ALP activity after 4 days, while 2 h exposure significantly increased mineralization after 14 days. Four-hour SS treatment increased OCN mRNA measured after 8 days and nuclear protein binding to an OSE2 site measured after 4 days. The calvarial defects showed slight bone healing in the control group. However, scaffolds adsorbed with low or high-dose oxysterol cocktail significantly enhanced bone formation. Histologic examination confirmed bone formation in the defect sites grafted with oxysterol-adsorbed scaffolds, compared to mostly fibrous tissue in control sites. Our results suggest that brief exposure to osteogenic oxysterols triggered events leading to osteoblastic cell differentiation and function in vitro and bone formation in vivo. These results identify oxysterols as potential agents in local and systemic enhancement of bone formation.

Use of ultra-high molecular weight polycaprolactone scaffolds for ACL reconstruction.

Previously, we reported on the implantation of electrospun polycaprolactone (PCL) grafts for use in ACL tissue engineering in a small animal model. In the present study, we hypothesized that grafts fabricated from ultra-high molecular weight polycaprolactone (UHMWPCL) would have similarly favorable biologic properties but superior mechanical properties as compared to grafts fabricated from PCL. Two forms of polycaprolactone were obtained (UHMWPCL, MW = 500 kD, and PCL, MW = 80 kD) and electrospun into scaffolds that were used to perform ACL reconstruction in 7-8 week old male Lewis rats. The following groups were examined: UHMWPCL, PCL, flexor digitorum longus (FDL) allograft, native ACL, as well as sham surgery in which the ACL was transsected. At 16 weeks post-operatively, biomechanical testing, histology, and immunohistochemistry (IHC) were performed. Analysis of cellularity indicated that there was no significant difference among the UHMWPCL, PCL, and FDL allograft groups. Quantification of birefringence from picrosirius red staining demonstrated significantly more aligned collagen fibers in the allograft than the PCL group, but no difference between the UHMWPCL and allograft groups. The peak load to failure of the UHMWPCL grafts was significantly higher than PCL, and not significantly different from FDL allograft. This in vivo study establishes the superiority of the higher molecular weight version of polycaprolactone over PCL as a scaffold material for ACL reconstruction. By 16 weeks after implantation, the UHMWPCL grafts were not significantly different from the FDL allografts in terms of cellularity, peak load to failure, stiffness, and collagen fiber alignment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:828-835, 2016.

Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL-derived cells.

There has been substantial effort directed toward the application of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue-specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)-derived cell populations with the potential for ligament regeneration: ligament-forming fibroblasts (LFF: CD146(neg) , CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ), ligament perivascular cells (LPC: CD146(pos) CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ) and ligament interstitial cells (LIC: CD34(pos) CD146(neg) , CD44(pos) , CD31(neg) , CD45(neg) )-and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8- and 3.3-fold, respectively) and LFFs (by 3- and 3.5-fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4- and 3.5-fold, respectively), LFFs (by 5.5- and 3-fold, respectively), LPCs (by 10- and 4.5-fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:985-994, 2016.

In vitro and in vivo evaluation of heparin mediated growth factor release from tissue-engineered constructs for anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) rupture is a common injury often necessitating surgical treatment with graft reconstruction. Due to limitations associated with current graft options, there is interest in a tissue-engineered substitute for use in ACL regeneration. While they represent an important step in translation to clinical practice, relatively few in vivo studies have been performed to evaluate tissue-engineered ACL grafts. In the present study, we immobilized heparin onto electrospun polycaprolactone scaffolds as a means of incorporating basic fibroblast growth factor (bFGF) onto the scaffold. In vitro, we demonstrated that human foreskin fibroblasts (HFFs) cultured on bFGF-coated scaffolds had significantly greater cell proliferation. In vivo, we implanted electrospun polycaprolactone grafts with and without bFGF into athymic rat knees. We analyzed the regenerated ACL using histological methods up to 16 weeks post-implantation. Hematoxylin and eosin staining demonstrated infiltration of the grafts with cells, and picrosirius red staining demonstrated aligned collagen fibers. At 16 weeks postop, mechanical testing of the grafts demonstrated that the grafts had approximately 30% the maximum load to failure of the native ACL. However, there were no significant differences observed between the graft groups with or without heparin-immobilized bFGF. While this study demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture, it also demonstrates that in vitro results do not always predict what will occur in vivo.

Athymic rat model for evaluation of engineered anterior cruciate ligament grafts.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Urinary bladder smooth muscle engineered from adipose stem cells and a three dimensional synthetic composite.

Human adipose stem cells were cultured in smooth muscle inductive media and seeded into synthetic bladder composites to tissue engineer bladder smooth muscle. 85:15 Poly-lactic-glycolic acid bladder dome composites were cast using an electropulled microfiber luminal surface combined with an outer porous sponge. Cell-seeded bladders expressed smooth muscle actin, myosin heavy chain, calponinin, and caldesmon via RT-PCR and immunoflourescence. Nude rats (n=45) underwent removal of half their bladder and repair using: (i) augmentation with the adipose stem cell-seeded composites, (ii) augmentation with a matched acellular composite, or (iii) suture closure. Animals were followed for 12 weeks post-implantation and bladders were explanted serially. Results showed that bladder capacity and compliance were maintained in the cell-seeded group throughout the 12 weeks, but deteriorated in the acellular scaffold group sequentially with time. Control animals repaired with sutures regained their baseline bladder capacities by week 12, demonstrating a long-term limitation of this model. Histological analysis of explanted materials demonstrated viable adipose stem cells and increasing smooth muscle mass in the cell-seeded scaffolds with time. Tissue bath stimulation demonstrated smooth muscle contraction of the seeded implants but not the acellular implants after 12 weeks in vivo. Our study demonstrates the feasibility and short term physical properties of bladder tissue engineered from adipose stem cells.